1. Field of the Invention
The present invention relates generally to a method of forming a two-dimensional distribution image of calcium ion concentration, hydrogen ion concentration, etc. in a cell, by use of a fluorescent microscope system in which a fluorescent reagent is employed as a probe. More particularly, this invention relates to a method of clearly displaying an image of a cell contour, and displaying the two-dimensional distribution image of the ion concentration in precise positional relationship with the cell contour.
2. Description of the Related Art
Recently, in the fields of neuroscience and cell biology, a method of determining the concentration of free calcium ions in a living cell has widely been employed to study the metabolic function of a cell. In this method, a suitable fluorescent reagent (hereinafter, referred to as "fluorescent probe") capable of functioning as a probe for assaying a target substance is introduced into a living cell. On the basis of the fluorescence intensity of the probe, the concentration of a target chemical species is determined. This method is carried out to assay various kinds of ions in a cell, such as calcium ions, sodium ions, magnesium ions, and chlorine ions, or macromolecules in a cell, such as protein or nucleic acid. The principle of assaying will now be explained.
When the fluorescent probe is bonded to a specific substance to be assayed, its fluorescence spectrum or excitation spectrum is changed. The degree of change differs in accordance with the concentration of the substance. Thus, if the fluorescence intensity of the probe is measured by means of a fluorescent spectrophotometer or a fluorescent microscope, the concentration of the specific substance can be determined on the basis of the change in the spectrum. For example, when calcium ions in a cell are determined, fura-2 is administered as a fluorescent probe, and the change in excitation spectrum caused by the specific bonding between the probe and calcium ions is measured. When a fluorescent microscope is used, it is noted that the change in fluorescence intensity is measured with respect to not the entire fluorescent image but a specific small circular region of the fluorescent image.
According to the above method, the concentration of calcium ions in a specific small region of a cell can easily been determined, but it is very difficult to find a concentration distribution of calcium ions in a relatively large two-dimensional region. In order to obtain such a concentration distribution, it is necessary to measure samples taken at many points, and a great deal of time and work is required. On the other hand, in the fields of neuroscience and cell biology, it is very important to find a distribution of calcium ion concentration in a relatively large region including, e.g. several cells. Under the situation, there is a demand for an improved method of easily obtaining a two-dimensional concentration distribution by measuring, at a time, the concentration of a target substance to be assayed in a relatively large two-dimensional region.
To meet such a demand, U.S. Pat. application Ser. No. 340,236, filed on Apr. 19, 1989 proposes a fluorescent microscope system combined with an image processing technique. The disclosure in said U.S. Pat. application which falls within the scope of this invention is incorporated in the present specification. The use of this fluorescent microscope system makes it possible to easily obtain an intracellular ion concentration distribution in a relatively large two-dimensional region. This type of fluorescent microscope is employed in a preferred embodiment of the present invention. Thus, the microscope will be described in detail in the description of the embodiment of the present invention.
Even if the above-stated fluorescent microscope system is employed, however, the following problems occur in the conventional method of obtaining a two-dimensional distribution of the intracellular ion concentration.
When the physiological function of a cell is studied, it is very important to obtain data as to in which portion of the cell the concentration of free calcium ions increases or decreases. However, a fluorescence intensity image obtained by the conventional method generally includes only data relating to an ion concentration, and does not include data relating to the positional relationship between the ion concentration and the cell. Thus, even if the obtained fluorescence intensity image is image-processed, it is not possible to obtain an image wherein the ion concentration distribution is related to the contour of the cell.
In addition, the obtained fluorescence intensity image includes background fluorescence outside the cell. In order to find a two-dimensional concentration distribution of ions to be assayed, the background fluorescence must be subtracted from the fluorescence intensity image. It is difficult, however, to determine the value of background fluorescence objectively. According to the conventional method, an experimenter determines the value of a background level empirically on the basis of a profile of a fluorescence intensity image. Thus, the basis for determining the value of a background level is uncertain, and varies from experimenter to experimenter. It is therefore difficult to precisely determine the ion concentration.